Conformational Dynamics in Designer Cellulosomes Studied by Single-Pair Fret with Mfd-Pie

Abstract

Cellulose is the most abundant biopolymer on earth and when broken down into oligosugars, it can be used for the production of bioethanol. As such, cellulose holds great potential as a novel energy source. The anaerobic thermophilic bacterium Clostridium thermocellum expresses an extracellular multi-enzyme complex that degrades cellulosose: the cellulosome. Cellulosomes, in general, consist of a scaffoldin domain harboring binding sites for multiple cellulases. These enzymes interact via their dockerin domain with complementary cohesin domain on the scaffoldin. An emerging research field aims at engineering ‘designer cellulosomes’ by genetically coupling different types of cohesins. These novel minimal cellulosomes should provide similar, if not better, enzymatic activity as the wild-type. Little is known about the exact role of the linker peptides between cohesin moieties. To address this, we have site-specifically labeled a designer cellulosome consisting of two cohesin subunits connected by either a natural or shortened peptide linker and performed single pair Forster resonance energy transfer experiments using pulsed interleaved excitation and multi-parameter fluorescence detection. We observe different conformational states of the cohesin dimer, suggesting the presence of conformational dynamics. Relating the conformational dynamics of such designer cellulosomes with their activity will be of great help for understanding and improving their function.